Yano T, Tokui T, Nishi Y, Nishizawa K, Shibata M, Kikuchi K, Tsuiki S, Yamauchi T, Inagaki M
Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Japan.
Eur J Biochem. 1991 Apr 23;197(2):281-90. doi: 10.1111/j.1432-1033.1991.tb15909.x.
Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.
角蛋白是上皮细胞中间丝的组成蛋白，是含有磷酸丝氨酸和磷酸苏氨酸的磷蛋白。我们研究了环磷酸腺苷（cAMP）依赖性蛋白激酶、蛋白激酶C和钙离子/钙调蛋白依赖性蛋白激酶II对角蛋白丝的体外磷酸化作用。当以1:1比例由I型角蛋白18（分子量47 kDa；酸性型）和II型角蛋白8（分子量55 kDa；碱性型）重构的大鼠肝脏角蛋白丝用作底物时，所有蛋白激酶均以显著的速率和程度使两种组成蛋白磷酸化，并且角蛋白丝结构发生解体。动力学分析表明，与角蛋白18相比，所有这些蛋白激酶优先使角蛋白8磷酸化。被cAMP依赖性蛋白激酶或蛋白激酶C磷酸化的角蛋白8和18的氨基酸残基几乎全是丝氨酸，而被钙离子/钙调蛋白依赖性蛋白激酶II磷酸化的是丝氨酸和苏氨酸。肽图谱分析表明，这些蛋白激酶以不同方式使角蛋白8和18磷酸化。这些观察结果为体内研究磷酸化在角蛋白丝重组中的作用开辟了道路。
